Recently, it has become clear that a long non-coding RNA (IncRNA) which does not encode a protein exerts important roles in various life phenomena and thus has attracted attention. In addition, it has also been known that the IncRNA, such as HOTAIR and Xist, will function by binding to a specific protein. From these facts, in analysis of the function of IncRNA, it is an important key to investigate what kind of protein binds to the target IncRNA, and forms a ribonucleoprotein complex (a complex of an RNA and an RNA-binding protein; a RNP complex).
In order to identify the protein which binds to the IncRNA and constitutes the RNP complex, it is necessary to isolate the RNP complex containing the target IncRNA from samples such as cells and tissues. As one of the methods, RNA pull-down assay has been known.
The RNA pull-down assay is a method comprising, synthesizing a biotin-labeled target RNA (for example, IncRNA) by in vitro transcription reaction, subjecting it to a reaction with a sample such as a cell lysate, and then isolating the protein (the RNA-binding protein) interacting with the target RNA as the RNP complex of the RNA-binding protein and the target RNA by using streptavidin beads.
This RNA pull-down assay is an effective technique for carrying out analysis research of the function of IncRNA, since it is capable of isolating the RNP complex, even for the IncRNA where the protein interacting with the RNA is unknown. However, there is a problem in the RNA pull-down assay that there are many cases making difficult to detect only the target RNA-binding protein specifically, because proteins other than the target RNA-binding protein present in the sample also bind nonspecifically to the streptavidin beads.
On the other hand, tamavidin (“TAMAVIDIN” is a registered trademark of Japan Tobacco Inc.) is a novel heat-resistant and avidin-like protein discovered by Japan Tobacco Inc., in an edible mushroom, Pleurotus cornucopiae. In addition, tamavidin 2 is a novel avidin-like protein having binding activity to biotin (WO 2002/072817: PATENT LITERATURE 1), similarly as avidin which is a basic protein derived from egg white, or streptavidin derived from Actinomycetes (Streptomyces avidinii). Further, tamavidin 2-REV has been developed, which is a modified version of tamavidin 2 obtained by modifying an amino acid sequence of tamavidin 2, and has such extent of binding force that a bonding can be dissociated, while maintaining binding force sufficient to bind to biotinylated substances (PATENT LITERATURE 2, NON-PATENT LITERATURE 1). Furthermore, it has been clarified, in carrying out purification of a biotinylated BSA from E. coli lysate using the tamavidin 2-REV immobilized-carrier (NON-PATENT LITERATURE 1), that the biotinylated BSA bound to tamavidin 2-REV immobilized-carrier can be eluted by the addition of a 0.1 M sodium phosphate buffer solution containing excess quantity of biotin (5 mM).
Under such circumstances, the present inventor has considered that a problem in the conventional RNA pull-down assay that proteins other than the target RNA-binding protein are contaminated because of nonspecific protein adsorption to the beads may be solved by carrying out the RNA pull-down assay using tamavidin 2-REV instead of streptavidin, and have carried out the study. As a result, the contamination of nonspecifically adsorbed proteins that causes high background was removed as much as possible, and thus [biotinylated RNA probe to which the RNA-binding protein is bound] could be obtained, when [the biotinylated RNA probe to which the RNA-binding protein is bound] was separated from [the complex of the RNA-binding protein, biotinylated RNA, and tamavidin 2-REV-immobilized magnetic beads] under a mild condition using an elution solution containing excess quantity of biotin (NON-PATENT LITERATURE 2).